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RESEARCH ARTICLE

Prolonged Subcutaneous Administration of Oxytocin Accelerates Angiotensin II-Induced Hypertension and Renal Damage in Male Rats James Phie1, Nagaraja Haleagrahara1, Patricia Newton1, Constantin Constantinoiu1, Zoltan Sarnyai1,2, Lisa Chilton1, Robert Kinobe1,2* 1 College of Public Health, Medical & Veterinary Sciences, James Cook University, Townsville, Queensland 4811 Australia, 2 Centre for Biodiscovery & Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Townsville Queensland 4811, Australia * [email protected]

Abstract OPEN ACCESS Citation: Phie J, Haleagrahara N, Newton P, Constantinoiu C, Sarnyai Z, Chilton L, et al. (2015) Prolonged Subcutaneous Administration of Oxytocin Accelerates Angiotensin II-Induced Hypertension and Renal Damage in Male Rats. PLoS ONE 10(9): e0138048. doi:10.1371/journal.pone.0138048 Editor: Jaap A. Joles, University Medical Center Utrecht, NETHERLANDS Received: May 14, 2015 Accepted: August 24, 2015 Published: September 22, 2015 Copyright: © 2015 Phie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data underlying the findings described in our manuscript are provided within the manuscript or as supplemental files and is fully available without restrictions.

Oxytocin and its receptor are synthesised in the heart and blood vessels but effects of chronic activation of this peripheral oxytocinergic system on cardiovascular function are not known. In acute studies, systemic administration of low dose oxytocin exerted a protective, preconditioning effect in experimental models of myocardial ischemia and infarction. In this study, we investigated the effects of chronic administration of low dose oxytocin following angiotensin II-induced hypertension, cardiac hypertrophy and renal damage. Angiotensin II (40 μg/Kg/h) only, oxytocin only (20 or 100 ng/Kg/h), or angiotensin II combined with oxytocin (20 or 100 ng/Kg/h) were infused subcutaneously in adult male Sprague-Dawley rats for 28 days. At day 7, oxytocin or angiotensin-II only did not change hemodynamic parameters, but animals that received a combination of oxytocin and angiotensin-II had significantly elevated systolic, diastolic and mean arterial pressure compared to controls (P < 0.01). Hemodynamic changes were accompanied by significant left ventricular cardiac hypertrophy and renal damage at day 28 in animals treated with angiotensin II (P < 0.05) or both oxytocin and angiotensin II, compared to controls (P < 0.01). Prolonged oxytocin administration did not affect plasma concentrations of renin and atrial natriuretic peptide, but was associated with the activation of calcium-dependent protein phosphatase calcineurin, a canonical signalling mechanism in pressure overload-induced cardiovascular disease. These data demonstrate that oxytocin accelerated angiotensin-II induced hypertension and end-organ renal damage, suggesting caution should be exercised in the chronic use of oxytocin in individuals with hypertension.

Funding: This work was supported by a research grant (2140-12005-0130 to RK & LC) from the Australian Institute of Tropical Health and Medicine, and James Cook University. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AngII, angiotensin II; ANP, atrial natriuretic peptide; ATP, adenosine triphosphate; HDOT, high dose oxytocin; LDOT, low dose oxytocin; NFAT, nuclear factor of activated T-cells; OT, oxytocin; SEM, standard error of the mean.

Introduction Recent advances indicate that oxytocin (OT) is potentially a useful therapeutic agent for many neuropsychiatric conditions including autism, social anxiety, postnatal depression, obsessivecompulsive problems and schizophrenia [1, 2]. However, the vast majority of studies on the putative use of OT as a therapeutic agent have been based on a wide range of single or shortterm OT doses in normal individuals and thus failed to determine how acute and chronic OT administration differ, and how the therapeutic effects of OT in normal individuals may translate to patients with comorbidities of brain disorders and other systemic disease conditions [3]. The oxytocinergic system influences physiological functions in many organ systems outside the central nervous system. OT is synthesised in the heart and the OT receptor similar to that found in the uterus and brain was identified in mammalian cardiac myocytes, cardiac fibroblasts and blood vessels [4, 5]. The first cardiac specific role of OT was demonstrated by its ability to trigger the release of atrial natriuretic peptide (ANP) from mammalian hearts [6], causing natriuresis and a decrease in blood pressure [7]. Similarly, OT is associated with a direct, centrally mediated regulation of autonomic outflow to the heart leading to negative inotropic and chronotropic effects [8, 9]. In experimental models of ischemic cardiac injury, single sub-pressor doses (25–125 ng/Kg) of OT significantly reduced myocardial infarct size, improving left ventricular function by activating cytoprotective intracellular mechanisms including the stimulation of endothelial nitric oxide synthase-guanylate cyclase, ANP-cyclic guanosine monophosphate, phosphoinositide 3-kinase and protein kinase B pathways likely via the OT receptor [10–12]. In the heart, these intracellular signalling mechanisms subserve many beneficial functions including vasodilation, anti-apoptotic and anti-fibrotic effects [10, 13]. It has also been suggested that the protective effects of OT in myocardial ischemic injury are linked to ischemic preconditioning [10, 11]. Acute stimulation of the cardiac OT receptor caused the up-regulation of protein kinase C, which triggers the activation of mitochondrial ATP-dependent potassium channels (mitoKATP) [10, 13]. This elicits cardiac protective effects through increased potassium ion uptake into the mitochondria, leading to reduced mitochondrial steady-state matrix volume, respiratory uncoupling and matrix alkalization [14]. Opening of mitoKATP channels also causes sustained production of reactive oxygen species during the ischemic period but reduces it following reperfusion thus preventing irreversible opening of the mitochondrial permeability transition pore [15]. In marked contrast however, the acute short-term and specifically targeted benefits of OT could be negated leading to unpredictable outcomes if the use of this neuropeptide is prolonged in chronic cardiovascular conditions such as hypertension and pressure overload-induced cardiac and renal damage. For instance, in ischemic cardiac injury, a short-term targeted administration of OT increased the expression of transforming growth factor-beta1 in the infarct and was associated with improved cardiac function and decreased mortality [11, 16, 17]. However, chronically increased transforming growth factor-beta 1 expression causes pathological cardiac remodelling characterised by hypertrophy, apoptosis and fibrosis [18]. This demonstrates that possible chronic effects of OT on cardiovascular function cannot be inferred from studies with acute dosing regimens. Furthermore, activation of the OT receptor is known to increase intracellular calcium concentration mediated by the stimulation of phospholipase C as seen in the myometrium [19, 20]. In the heart, accumulation of intracellular calcium and the subsequent activation of calcium dependent calcineurin activity, and its downstream effector, the nuclear factor of activated T-cells (NFAT) is a hallmark and a critical intracellular signalling convergence point for many pathological conditions, including chronic pressure overload-induced cardiac hypertrophy and failure, and ischemic injury [21, 22]. Mammalian myometrial cells and cardiac myocytes share an identical OT receptor [23] but OT treatment appears to

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ameliorate endothelin-1 and Angiotensin II (AngII) mediated NFAT activation, hypertrophy and apoptosis in neonatal rat cardiac myocytes [24]. While this in vitro study based on neonatal cardiac myocytes indicates that OT may attenuate the effects of pressure-overload induced cardiac hypertrophy and pathology, effects of prolonged OT administration on cardiovascular health in general, and chronic pressure overload-induced cardiac hypertrophy and end-organ damage in vivo have not been investigated. We hypothesised that in the presence of a chronic hypertensive state, prolonged administration of OT may be pathological. Our objective was to examine effects and putative pathophysiological mechanisms of prolonged administration of low dose OT in a rodent model of AngII-induced hypertension, cardiac hypertrophy and renal damage.

Materials and Methods Chemicals and Reagents OT was obtained from Bachem (Bubendorf, Switzerland), AngII and all other chemicals were obtained from Sigma Chemical Company (St. Louis, MO USA). Bioassay reagents for quantification of total plasma renin and ANP, and a bioassay kit for extraction and quantification of myocardial tissue calcium dependent calcineurin activity were obtained from Sapphire Biosciences (NSW, Australia). Implantable mini-osmotic pumps (Model 2004) were obtained from Alzet (CA, USA).

Animals and experimental design Male Sprague Dawley rats (8–9 weeks old, 240–350g) were used for this study. Rats were housed (2–3 animals per cage), at 23°C, 60% humidity on a 14:10 hour light-dark cycle; were fed standard rat chow and allowed access to water ad libitum. Animal use and all experimental procedures were approved by James Cook University Animal Ethics Committee according to Australian guidelines for the use and care of laboratory animals (Approval number A2010). Forty-eight rats were randomly divided into six experimental groups (n = 8 per group) and starting body weights were recorded (S1 File). The experimental groups consisted of: Control receiving phosphate buffered saline; Low dose OT (LDOT, 20 ng/Kg/h); High dose OT (HDOT, 100 ng/Kg/h); AngII (40 μg/Kg/h); AngII with LOT and AngII with HDOT. The dose of AngII was based on that used in previous models of hypertension and cardiac hypertrophy induced by AngII infusion [25, 26]. The doses of OT were based on those used in acute studies utilising OT infusion in myocardial infarction [11]. AngII and OT were dissolved in phosphate buffered saline and treatments were administered for 28 days via subcutaneously implanted mini-osmotic pumps. One animal in the AngII only group died suddenly of an unidentified cause before the conclusion of experiments but this did not warrant any changes in the experimental design.

Osmotic pump implantation Mini-osmotic pumps were prepared as per manufacturer's instructions. Rats were anaesthetised with isoflurane (5% induction, 2% maintenance, at a flow rate of 2 L/min in oxygen) and an incision approximately 1 cm in diameter was made between the scapulae and pumps were inserted into the incision. Once surgery was completed, wounds were closed using 5.8 mm surgical staples and swabbed with 7.5% w/v iodine to prevent contamination and infection.

Hemodynamic measurements Baseline diastolic, systolic and mean arterial blood pressures, and heart rate were measured immediately prior to pump implantation and at day 7 and day 28 after commencement of the

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experiment. Animals were acclimatized to the restraint apparatus on a heated pad at 37.5°C in a quiet environment under light isoflurane-induced anaesthesia (4% induction, 2% maintenance, at a flow rate of 2 L/min in oxygen). Measurements were taken using a computerised CODA blood pressure monitor (Kent Scientific, CT USA), equipped with an occlusion cuff and a volume pressure recording sensor. Values were recorded until five complete measurements were obtained without the range of the five replicate mean arterial pressures exceeding 15mmHg. All blood pressure measurements were taken between the hours of 6:00 AM-11:00 AM to minimise the impact of diurnal variation. This method has been validated and shown to have good reproducibility and accuracy over the physiological range of blood pressure values in rodents [27].

Echocardiography Echocardiograms (M-mode) were obtained 21 days after pump implantation (n = 7–8 per group), under light isoflurane-induced anaesthesia as described above. Rats were positioned right side down on a heated pad and echocardiograms were obtained via the right parasternal short axis view just below the level of the mitral valve using a curved array (3–9 Mhz) probe on 1 a MyLab70 XVision Esaote ultrasound machine. Left ventricular mass was calculated using the American Society of Echocardiography formula in Eq (1) below and then corrected for variations in body weight where 1.04 is the specific gravity of muscle, LVIDd is the left ventricular internal diameter in diastole, IVSd is the interventricular septal diameter in diastole and LVPWd is the left ventricular posterior wall thickness in diastole. 3

3

1:04½ðIVSd þ LVIDd þ LVPWd Þ  ðLVIDd Þ 

Eqð1Þ

Fractional shortening (FS) was calculated according to Eq (2), where LVIDS is the left ventricular internal diameter in systole and LVIDd is the left ventricular internal diameter in diastole. ðLVIDd  LVIDs Þ =ðLVIDd Þ  100

Eqð2Þ

For each measured parameter, an average of five cardiac cycles was used by a veterinary echocardiologist who was blinded to the experimental treatment regimens.

Histological and morphometric analysis of cardiac hypertrophy and renal damage At day 28, animals were sacrificed by carbon dioxide asphyxiation. Hearts were fixed in diastole by a retrograde apical infusion of 1% potassium chloride, rapidly removed, blot dried, weighed and cut in half along the cross sectional plane. The basal half of the heart and the left kidney were fixed in 10% neutral buffered formalin and embedded in paraffin for histological analysis. Sections of the heart and kidney (5μm) were prepared and stained with hematoxylin and eosin, and Masson’s trichrome, and then photographed under a bright-field with a microscope (Olympus BX43F). Collagen deposition in the posterior wall of the left ventricle and the interventricular septal wall was analysed using Image-J software (NIHealth, MD USA), according to previously described methods [28]. For kidney sections, a histological scoring system based on a semi-quantitative analysis of pathological changes [29] was also used to assess AngII or OT induced changes in a single-blinded manner. Assessed parameters included: glomerular necrosis, tubular degeneration, necrosis and epithelial sloughing, vascular congestion and extravasation, and interstitial fibrosis. The grading system used to assess these pathological changes was based on 0–4 score designed by a pathologist who was blinded to the treatments and was outlined as, 0 = normal with no noticeable histopathological changes; 1 = minor: 0–29%;

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2 = moderate: 30–49%; 3 = marked: 50–69% and 4 = severe: 70–100%. Six individual samples were analysed for each of the six experimental groups and histological scores were reported as mean ± standard error of the mean (SEM).

Quantifying plasma electrolytes, creatinine, urea, ANP and prorenin/ renin At day 28, rats were euthanized by carbon dioxide overdose, blood was collected in heparinised tubes and plasma samples (n = 6 per experimental group) were collected by centrifugation at 2000 xg, for 15 min at 4°C and then stored at -20°C prior to analysis. Photometric analysis was performed to determine plasma sodium, potassium, chloride, calcium, magnesium, phosphate, bicarbonate, urea, creatinine, total protein and glutamate dehydrogenase activity using a chemistry analyser (Beckman, AU480). Urea to creatinine ratio was calculated as an indicator for renal failure. Urine flow rate was not measured in this study but an indirect measure of creatinine clearance (mL/min) was used according to a method that utilises plasma creatinine concentration and body mass index in rats [30], using Eq (3). CrðmL = minÞ ¼ 220  103 ðmmol =min = kgÞ  weightðkgÞ  ½Crðmmol =LÞ

1

ð3Þ

Where Cr is plasma creatinine concentration. Total plasma prorenin/renin concentration was determined using an ELISA assay kit, according to the manufacturer’s instructions (Molecular Innovations) and plasma ANP was quantified by using an ANP ELISA kit [31].

Calcineurin activity Frozen heart tissue was thawed and a section of the left ventricle (0.20–0.35g) was isolated and homogenised using a Polytron (Brinkmann PT2100) tissue homogeniser in lysis buffer containing 50 mmol/L Tris (pH 7.5), 0.1 mmol/L EDTA, 0.1 mmol/ L EGTA, 0.2% NP-40, 1 mmol/ L DTT, 100 μmol/ L PMSF, 5 mmol/L ascorbic acid, and protease inhibitor cocktail. The tissue homogenate was centrifuged at 18000 xg for 30 min at 4°C and the supernatant was desalted to remove excess phosphate and nucleotides. Calcineurin activity was measured by the rate of dephosphorylation of a synthetic peptide using a calcineurin assay kit according to the manufacturer’s instructions. The released free phosphate was detected using the standard Malachite green colorimetric assay [32]. Relative calcineurin phosphatase activity was expressed as pmol phosphate/μg of protein.

Statistical Analysis Normally distributed data were presented as means ± SEM and analysed using one-way analysis of variance with Tukey's post-hoc tests for multiple comparisons. Data that were not consistent with a Gaussian distribution were presented as medians with the interquartile range (IQR) and analysed using the Kruskal-Wallis test paired with Dunn’s post-hoc test for multiple comparisons and the linear mixed effects model to adjust for unequal variance between experimental groups [33]. Changes were considered significant at P < 0.05.

Results Chronic Administration of Oxytocin Accelerated Angiotensin II-induced Hypertension Blood pressure and heart rate was measured immediately before pump implantation (day 1) and after 7 and 28 days of OT, AngII or AngII and OT infusion. Administration of only OT at

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Fig 1. Effects of oxytocin and angiotensin II on systolic and diastolic blood pressure. Systolic and diastolic blood pressure (mmHg) after 1, 7 and 28 days of subcutaneous infusion of saline (control), low dose oxytocin (LDOT), high dose oxytocin (HDOT), angiotensin II (AngII), a combination of angiotensin II and low dose oxytocin (AngII + LDOT) or angiotensin II and high dose oxytocin (AngII + HDOT). Data represent medians with interquartile range (n = 5–8). Asterisks (*) indicate significant difference from the control group on the same day (* P